PROJECT SUMMARY The opportunistic fungus Cryptococcus neoformans causes a meningoencephalitis that kills hundreds of thousands of people each year worldwide. The long-term goal of our research is to understand the pathogenesis of this devastating disease. This application focuses on a central aspect of cryptococcal infection, the interactions of this facultative intracellular pathogen with the phagocytic cells of its mammalian host. These interactions determine the outcome of infection, yet major gaps remain in our understanding of how they occur and influence downstream events. We propose a genome-wide strategy to discover C. neoformans factors that mediate or influence these interactions, and to explore a subset of them. In Aim 1 we propose to identify, on a whole-genome scale, mutations that cause altered fungal uptake by primary host cells. We will do this by using a powerful image-based assay to screen a newly available C. neoformans deletion collection. Deletion strains that show significant phenotypic alterations with both mouse and human primary cells will advance to Aim 2, in which we propose to test mutants in a mouse model of infection, construct strains to confirm and complement mutant phenotypes, and assess fungal fate within host cells. Together, these studies will yield a prioritized group of mutants that demonstrate aberrant interactions with both mouse and human phagocytes and also exhibit altered virulence in a mouse model. This information will serve as a springboard to future mechanistic studies in this critical area of fungal pathogenesis. Our strong relevant expertise makes it feasible to perform rigorous studies on a whole genome scale in the timeframe of an R21 application. Completing the proposed experiments will identify cryptococcal factors that influence crucial events of pathogenesis, either by directly mediating interactions with host cells or by indirectly influencing them. This exciting step forward in knowledge will position us and other investigators to pursue these factors mechanistically. It will likely lead to the discovery of novel factors that influence pathogenesis, open new directions for the study of C. neoformans and other microbial pathogens, and potentially suggest points of intervention to advance antifungal therapy and improve the outcome of a devastating disease.